Structures and structure-activity relationships of three mitogenic and complement fixing pectic arabinogalactans from the malian antiulcer plants Cochlospermum tinctorium A. Rich and Vernonia kotschyana Sch. Bip. ex Walp.

Structures of three pectic arabinogalactans, one from Vernonia kotschyana (Vk2a) and two from Cochlospermum tinctorium (Ct50A1 and Ct50A2), and their complement fixation and induction of B cell proliferation in vitro were compared. The polysaccharide Vk2a expressed potent biological activity in both assays compared with Ct50A1 and Ct50A2. Vk2a possessed a very high molecular weight (1150 +/- 20 kDa) compared with Ct50A1 and Ct50A2 which both showed a polydisperse nature with the highest molecular weight polymers in each fraction estimated at approximately 105 kDa (Ct1a) and 640 +/- 100 kDa (Ct2a), respectively. The HMW polymers showed complement fixation in the same range as the native fractions. The arabinogalactan II content was low in Vk2a (2%) compared with that in Ct50A1 (23%) and Ct50A2 (12%). The high molecular weight polymers were subjected to digestion with a beta-d-(1, 3)-galactanase-rich fraction from Driselase, oligomers were isolated by HPAEC, and their finer structures were determined by MALDI- and ES-qoToF-MS, linkage, and monosaccharide composition analyses. Vk2a consists of both a galacturonan core and a rhamnogalacturonan core rich in neutral side chains. The backbones of both Ct-polysaccharides consist mainly of RG-I regions with numerous neutral side chains dominated by galactosyl residues, whereas the homogalacturonan regions seem to be small. Differences in the chain lengths of the 6-linked galacto-oligosaccharides attached to the 3-linked galactan core could not be related to the differences in the potencies of the biological activities observed.

Isolation of an N-acetyl-D-glucosamine specific lectin from the rhizomes of Arundo donax with antiproliferative activity.

A lectin with antiproliferative activity towards human cancer cell lines and mitogenic towards human peripheral blood mononuclear cells was purified from the rhizomes of Arundo donax (Linn.) by affinity chromatography on N-acetyl-d-glucosamine linked to epoxy-activated sepharose-6B. The pure preparation apparently yielded a single band of approximately 15 kDa on SDS-PAGE, pH 8.3, under both reducing and non-reducing conditions. The molecular mass of native lectin was 32 kDa as determined by gel filtration chromatography. This showed the lectin to be a dimer, with subunits not held together by disulphide linkages. The A. donax lectin (ADL) agglutinated rabbit erythrocytes and the agglutination was inhibited by N-acetyl-d-glucosamine and its di- and trimer. The lectin was thermostable upto 55 degrees C and showed optimum activity in the range of pH 7.0-9.0 and comprised of 2.1% carbohydrate content.

Isolation of two plant proteinases in latex from Carica candamarcensis acting as mitogens for mammalian cells.

In a prior study we showed evidence that latex from Carica candamarcensis contains a protein fraction that stimulates mammalian cell proliferation. In this report we describe the isolation of two proteinases responsible for this effect. Both proteinases (P1, P2) display a relative mass of 23 kDa and following chromatographic purification stimulate proliferation of fibroblastic and epithelial cells. P2 added to L929 fibroblasts at 2.5 nM enhances proliferation by 60 %. We further demonstrate that its cellular effect is linked to an increase in activity of Erk2, a component of the MAP kinase pathway. To our knowledge, this is the first known plant proteinase to exert a proliferative effect in mammalian cells. This novel mitogenic property attributed to a purified cysteine proteinase may explain some of the therapeutic actions attributed to these enzymes.

Purification of chrysancorin, a novel antifungal protein with mitogenic activity from garland chrysanthemum seeds.

A novel antifungal protein, designated chrysancorin, was isolated from seeds of Chrysanthemum coronarium var. spatiosum with a procedure involving ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue resin, ion exchange chromatography on SP-Sepharose and FPLC-gel filtration on Superdex 75. The N-terminus of chrysancorin displays sequence similarity to the genomic sequence of chromosome 1 from Arabidopsis thaliana BAC T19E23. Chrysancorin exhibits a molecular mass of 13.4 kDa in gel filtration and SDS-polyacrylamide gel electrophoresis. It stimulates the proliferation of mouse splenocytes and inhibits the activity of human immunodeficiency virus-1 reverse transcriptase. The protein possesses antifungal activity against Botrytis cinerea, Mycosphaerella arachidicola and Physalospora piricola, but not against Rhizoctonia solani, Fusarium oxysporum and Coprinus comatus. However, we could not detect antibacterial activity against a variety of bacteria.

Wide range of molecular weight distribution of mitogenic substance(s) in the hot water extract of a Chinese herbal medicine, Bupleurum chinense.

In this study, we examined the contribution of lignin-like materials in lower molecular weight (MW) fractions from the hot water extract of Bupleuri Radix (Bupleurum chinense) (HWE-BR) for their immunopharmacological activities. Mitogenic activity was detected in all the fractions of MW ranges: lower than 1.0 kDa, 1.0-3.5 kDa, 3.5-10 kDa, and 10-50 kDa. After NaClO2 treatment of these subfractions, UV spectra, ESR spectra, mitogenic activities on murine B-cells, and the activity of inducing nitric oxide in RAW 264.7 cells were significantly reduced, suggesting that lignin-like polyphenolic substance(s) of various MW might take part in these activities. The intensity of ESR spectra and mitogenic activities were stronger in higher MW subfractions, thus the content of stable radical species and/or the degrees of polymerization would be important for their immunopharmacological activities.

Immunomodulatory and antitumoral effects of triterpenoid saponins.

Genuine saponins of Solidago virgaurea L., Heteropappus altaicus (Willd.) Novopokr., Heteropappus biennis (Ldb.) Tamamsch. and Helianthus annuus L. (Asteraceae) as well as related carbohydrate modified glycosides of polygalacic acid and echinocystic acid and some commercial available triterpenoid saponins were investigated in view of their immunomodulating and antitumoral effects. Mitogeneic effects on murine spleen and thymus cells, as well on human mononuclear cells in vitro could be demonstrated. The activity of murine bone marrow macrophages was stimulated in a chemoluminescence assay, and further an induction of cytotoxic macrophages and a TNF alpha release from murine macrophages was observed. The mitogeneic and TNF alpha releasing virgaureasaponin E (1) showed in vivo antitumoral effects in the allogeneic sarcoma 180 tumor model and in the syngeneic DBA/2-MC.SC-1 fibrosarcoma tumor model. In mice, treated with 1, phagocytosis of bone marrow cells and proliferation of spleen and bone marrow cells were stimulated in an ex vivo assay whereby the TNF alpha concentration in blood considerably increased compared to the control group.

Mitogenic activities in African traditional herbal medicines.

Mitogenic activities in African traditional herbal medicines were examined using protein fractions obtained from their extracts by precipitation with ammonium sulfate. Potent mitogenic activities for human and mouse lymphocytes were found in the three plants: Croton macrostachyus, Croton megalocarpus (Euphorbiaceae), and Phytolacca dodecandra (Phytolaccaceae). All the gel chromatographic patterns of these protein fractions progressed toward the smaller molecule site with pronase treatment, while their mitogenic activities decreased significantly. Protein fractions from these three plants induced mitogenesis both in human and mouse isolated T cells, but not in lymphocytes from athymic nude mice. By testing further fractionated protein fractions with gel filtration chromatography, it was found that all three plants contained several mitogens having different molecule sizes.

Chemical structure and biological activity of polysaccharides from Hibiscus sabdariffa.

Three water-soluble polysaccharides have been isolated from flower buds of Hibiscus sabdariffa L. (HIB 1,2,3). The neutral polysaccharides (HIB 1 and 2) are composed of arabinans and arabinogalactans of low relative molecular mass. The major fraction was investigated by methylation analysis, pectinase-treatment, mild acid hydrolysis and NMR studies, and it was shown to be a pectin-like molecule (Mr = 10(5)d). The main chain is composed of alpha-1,4-linked GalA (24% methyl-esterified) and alpha-1,2-linked Rha. Side chains are built of Gal and Ara and are connected to the main chain via C-4 of every third Rha. Its structure seems to be different from polysaccharide structures described in other species of the Hibiscus genus and the Malvaceae family. All fractions were assayed for possible immune-modulating effects. All fractions showed some activity, but the main acidic fraction was contaminated with lipopolysaccharide, and therefore its shown activity has to be discussed carefully.

Heterogeneity and characterisation of mitogenic and anti-complementary pectic polysaccharides from the roots of Glycyrrhiza uralensis Fisch et D.C.

Two anti-complementary polysaccharide fractions (GR-2IIa and GR-2IIb), isolated from the roots of Glycyrrhiza uralensis Fisch et D.C., each gave five anti-complementary polysaccharides (GR-2IIa-1-5 and GR-2IIb-1-5) on h.p.l.c.; likewise, GR-2IIc gave two anti-complementary and mitogenic polysaccharides (GR-2IIc-1-2A and -2IIc-2) by gel filtration and h.p.l.c. GR-2IIc-1-2A showed the most potent anti-complementary activity. GR-2IIa-1-5 and GR-2IIb-1-5 contained 40-85% and 50-90% of GalA, respectively, in addition to Rha, Ara, and Gal. GR-2IIc-1-2A and -2IIc-2 mainly comprised Glc, Gal, GalA, and GlcA in addition to Rha, Fuc, Xyl, Ara, and Man. Methylation analysis and digestion with endo-alpha-(1----4)-polygalacturonase indicated that all of the polysaccharides contained polygalacturonan regions which were frequently methyl-esterified. GR-2II-a, -2IIb, and -2IIc gave enzyme-resistant fractions of large and intermediate sizes, in addition to oligogalacturonides. Each large fraction from GR-2IIa and -2IIb consisted mainly of Ara, Gal, and GalA, whereas the intermediate fractions were composed of small proportions of 2-Me-Fuc, 2-Me-Xyl, and apiose (Api), in addition to Rha, Ara, Gal, and GalA. The large fraction from GR-2IIc mainly contained Rha, Man, Gal, and GalA in addition to Fuc, Ara, Xyl, and Glc, whereas the intermediate fraction consisted of 2-Me-Fuc, 2-Me-Xyl, and Api, in addition to Rha, Ara, GalA, Fuc, Xyl, Man, Gal, and Glc. Base-catalysed beta-elimination followed by ethylation indicated that all the polysaccharides except GR-2IIc-2 contained a 4-linked uronic acid attached to position 2 of 2,4-linked Rha. Single radial gel diffusion, using the beta-D-glucosyl-Yariv antigen, indicated that GR-2IIa-1 and GR-2IIc-2 contained relatively large proportions of (1----3,6)-beta-D-galactan moieties. The anti-complementary activities of GR-2IIa-3, GR-2IIa-4, and GR-2IIb-4 decreased after de-esterification followed by digestion with endo-alpha-(1----4)-polygalacturonase. The large fractions from GR-2IIa-2IIc showed more potent anti-complementary activities than the original polysaccharide fractions, whereas the intermediate fractions and oligogalacturonides were inactive. The large fraction from GR-2IIc had more potent mitogenic activity than GR-2IIc, whereas the intermediate fraction and oligogalacturonides from GR-2IIc were inactive.

Characterization of mitogenic pectic polysaccharides from kampo (Japanese herbal) medicine "juzen-taiho-to".

The acidic polysaccharide fraction (F-5-2) from "Juzen-Taiho-To" (TJ-48), a Kampo (Japanese herbal) medicine prepared by decocting a prescription of 10 kinds of herbs, has potent mitogenic activity. In order to clarify the mitogenic activity, F-5-2 was fractionated by amon-exchange chromatography, and 14 acidic polysaccharide fractions were obtained. Mitogenic activities of these polysaccharides were increasing with their molecular masses and affinities to the anion-exchange column. Methylation analysis and endo-alpha-(1----4)-polygalacturonase digestion showed these polysaccharide fractions to be pectic polysaccharides, many of which consisted mainly of polygalacturonan regions in addition to small amounts of "ramified" regions. Gel filtration showed that the molecular masses of the "ramified" regions of the polysaccharides were similar. High molecular mass polygalacturonic acid showed a weak mitogenic activity. The mitogenic (M-1 and M-2) and non-mitogenic (M-3) polysaccharides from the most active polysaccharide fraction (F-5-2IIh) were further purified by gel filtration. M-2 and M-3 were also shown to be pectic polysaccharides, and the neutral glycosidic linkages in M-2 and M-3 were different from each other. Endopolygalacturonase digestion markedly decreased the mitogenic activity of M-2, however, the "ramified" region of M-2 still showed a weak activity.